Deregulation of the Cyclin D1/p16ink4a/pRB pathway is one of the most common defects in human cancers. The genes defining this pathway have been characterized as G1-specific cell cycle regulators, and their oncogenic properties attributed to this function. Drosophila Cyclin D kinase (CycD/Cdk4) is not a specific G1/S regulator. Rather, it promotes cell mass accumulation (growth), and this in turn promotes cell proliferation in some contexts. We propose several parallel approaches for identifying critical growth regulatory targets of CycD/Cdk4 in Drosophila. Difference gel electrophoresis (DIGE) will be used to map alterations in cellular protein composition (such as protein phosphorylation) caused by overexpressed CycD/Cdk4. A highly specific technique using ATP analogs will be employed to isolate in vivo substrates of CycD/Cdk4. Genes whose transcription is affected in vivo by overexpressed CycD/Cdk4 will be identified using DNA microarrays representing approximately 9000 Drosophila mRNAs. Rapid F1 genetic screens will identify genes that suppress or enhance hypertrophy of the Drosophila eye caused by ectopic CycD/Cdk4. These studies should identify genes and proteins that are substrates, activators, or suppressors of CycD/Cdk4. Following target identification, molecular and genetic methods will be used to characterize interactions of the targets with CycD/Cdk4, and test their functions in cell growth in vivo in Drosophila. These studies should reveal how CycD/Cdk4 alters cell physiology to promote growth, and provide a molecular paradigm explaining how cell growth and cell division are coupled. The identification of growth regulatory genes in flies should also provide entry points for studies of cell growth control in humans during both normal and neoplastic development.